Our finding that airway smooth muscle operates over a broad length range suggests 3 related hypotheses: I. Smooth muscle adapts to longer lengths by increasing the number of contractile elements in series. II. Thick-filament lengthening produces the well-known velocity slowing during the rise of activation. III. A thin-filament regulatory mechanism selectively engages filamentous myosin. We have obtained much evidence supporting all three hypotheses, leading to two Specific Aims: Specific Aim 1. Determine the function of the thin-filament regulatory proteins in airway smooth muscle. The dual role of myosin light-chain phosphorylation, promoting thick-filament formation and activating myosin's interaction with actin, suggests that another mechanism exists to prevent phosphorylated myosin monomers from interacting with actin before they join filaments. The observation that smooth muscle has thin-filament regulatory proteins similar to those that confer calcium sensitivity on striated muscle suggest that these proteins might provide the necessary selectivity of thin filaments for myosin in filaments and produce a calcium sensitivity similar to that in striated muscle. To test this suggestion, muscles will be permeabilzed with _-toxin to produce small holes in the surface membrane that allow the passage of small ions and ATP, but retain myosin light-chain kinase in the cells. They will then be permanently thiohosphorylated with ATP-_-S and tested to see if they can be contracted and relaxed with Calcium and EGTA. A positive result would accomplish Aim I and produce a useful tool for Aim II. Specific Aim2. Determine the relative contributions of factors that lengthen airway smooth muscle lattice. Our experiments have shown that airway smooth muscle adapts to longer lengths by placing more thick filaments in series, but that filament mass increases in only a 2:3 proportion with muscle length. Aim 2 is to discover the other factors that lengthen the lattice. To this end, mean thick-filament length and mean longitudinal distance between thick filaments will be measured in electron micrographs of muscles fixed at lengths that vary by 2-fold. In addition, when a means of repetitively activating and relaxing permeabilized muscles with fixed filament lattices, the muscles will be repetitively activated and relaxed at different lengths to determine the amount that filament sliding contributes to the length changes.